Yersinia pestis is the causative agent of plague, a disease that affects many different mammalian species. Plague is transmitted by fleas, which are infected during blood meals and colonized via a gastrointestinal biofilm. Transmission occurs when colonized insects feed on new hosts, typically rodents. Historically, epizootic outbreaks in rodents have triggered flea-born transmission of plague into human populations with devastating consequences. For this reason, Y. pestis is considered the single most virulent bacterial pathogen and weapon of mass destruction. Y. pestis is a clonal pathogen with a genome of 4,012 chromosomal genes and three virulence plasmids. The pCDI plasmid encodes the type III secretion machine for delivery of Yop effectors into host immune cells. This essential virulence strategy is regulated by two environmental signals: temperature shift, as bacteria are transferred from fleas (18-30C) to mammals (37C), and a drop in extracellular calcium ions, perceived as type III needle complexes penetrate host immune cells (LCR, low-calcium response). The functions of most genes on the virulence plasmids have been revealed, however very little is known about chromosomal genes and their contributions to the unique lifecycle of Y. pestis. The transcriptome of Y. pestis grown in vitro has been compared with bacteria in a flea biofilm and Y. pestis invading a rat bubo: 214 genes are specifically upregulated in the flea gut, whereas 119 genes are specifically expressed in the rat bubo. Here we will investigate the function of uncharacterized Y. pestis genes upregulated in either the flea gut or the rat bubo. Towards this goal, we have isolated 7,832 mutants with mapped insertional lesions on the Y. pestis C092 chromosome (PEST-Library). Mutants with lesions in unknown ORFs, hypothetical genes and small RNAs will be analyzed for defects in flea colonization, the LCR, type 111 secretion and effector translocation into host immune cells. Selected mutants will be analyzed for the pathogenesis of bubonic plague and the histopathological features of this disease. Mutants with defects in flea biofilm or plague pathogenesis will be subjected to biochemical studies, thereby exploring the specific functions of unknown gene products and small RNAs in the Y. pestis lifecycle.